小鼠仙台病毒HN蛋白单克隆抗体的制备及鉴定

Preparation and ldentification of monoclonal antibodies against mouse Sendai virus HN protein

为了制备小鼠仙台病毒(Sendai virus,SeV)HN蛋白单克隆抗体,试验采用SeV参考毒株HN基因(GenBank登录号为NC_001552.1)对编码HN蛋白的基因密码子优化后克隆重组至原核表达载体pGEX-6P-1上获得重组质粒pGEX-6P-1-HN,将其转化至大肠杆菌BL21感受态细胞中,用IPTG进行诱导表达,并对纯化的重组蛋白rHN进行SDS-PAGE分析。用重组蛋白rHN免疫Balb/c小鼠6次,取其脾脏细胞与小鼠骨髓瘤细胞SP2/0进行融合,通过亚克隆技术和间接ELISA方法筛选阳性杂交瘤细胞,然后注入小鼠腹腔制备抗HN蛋白单克隆抗体,分析单克隆抗体的免疫活性、特异性和稳定性。结果表明:获得3株分泌抗SeV HN蛋白抗体的杂交瘤细胞2F9、5D8、10H2,分泌的抗体效价均为1∶16 000左右。3株单克隆抗体的亚型均为IgG1,均可特异性识别SeV,并不与其他小鼠病毒产生交叉反应,具有良好的特异性。经过10次传代,3株单克隆抗体的效价均在1∶8 000以上,稳定性较好。说明试验成功制备了具备良好免疫活性、特异性和稳定性的抗SeV HN蛋白单克隆抗体,可以为SeV的检测提供有效试剂。

In order to prepare monoclonal antibodies against mouse Sendai virus(SeV) HN protein,in the experiment,the SeV reference strain HN gene(GenBank accession number NC_001552.1) was used,and the codon of the gene encoding the HN protein was optimized and then cloned and recombined into the prokaryotic expression vector pGEX-6P-1 to obtain the recombinant plasmid pGEX-6P-1-HN;it was transformed into Escherichia coli BL21 competent cells,and induced to be expressed by IPTG;the SDS-PAGE analysis was performed to purify the recombinant protein rHN.The recombinant protein rHN was used to immunize Balb/c mice for 6 times;the spleen cells were taken and fused with mouse myeloma cells SP2/0;the positive hybridoma cells were screened by subcloning and indirect ELISA,which were then injected into the peritoneal cavity of the mice to prepare the monoclonal antibodies against the HN protein;the immunoreactivity,specificity and stability of the monoclonal antibodies were analyzed.The results showed that three strains of hybridoma cells 2F9,5D8 and 10H2 secreting anti-SeV HN protein antibodies were obtained,and the titer of the secreted antibodies was about 1∶16 000.The subtypes of these three monoclonal antibodies were all IgG1 subtypes,and all of them could specifically recognize SeV and did not cross-react with other mouse viruses with good specificity.After 10 passages,the titer of the three monoclonal antibodies could be maintained above 1∶8 000 with good stability.The results suggested that the anti-SeV HN protein monoclonal antibodies with good immunogenicity,specificity and stability had been successfully prepared,which could provide effective reagents for the detection of SeV.